Determination and characterization of total thiols in mouse serum samples using hydrophilic interaction liquid chromatography with fluorescence detection and mass spectrometry

Abstract

Biothiols such as homocysteine, cysteine, and glutathione play many biologically important roles, especially in reduction-oxidation homeostasis and resistance to oxidative stress, and the measurement of their concentrations in model animal fluids is important in clarifying the pathology of thiol-related diseases. In this study, an analytical method for total biothiols in mouse serum using hydrophilic interaction liquid chromatography (HILIC) with fluorescence detection was developed. Mouse serum samples were derivatized with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F), after reduction by tris(2-carboxyethyl)phosphine. Five biothiols (homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine) in the mouse sera were separated within 16 min on an amide-type HILIC column. The method possessed good linearity, good reproducibility with an intra-day variance of less than 3%, and low detection limits of 0.2-4 nM. Concentrations of homocysteine, cysteine, cysteinylglycine, glutathione, and γ-glutamylcysteine in the mouse serum samples were calculated as 6.7 ± 0.3, 227.7 ± 16.9, 1.2 ± 0.4, 77.5 ± 29.2, and 8.2 ± 0.9 μM, respectively (mean ± S.D., n = 4). Furthermore, HILIC-negative electrospray ionization-mass spectrometry (MS) analysis using a high-resolution mass spectrometer was conducted to determine the exact masses of two unknown peaks, which were found in the mouse serum samples with high signal intensity and were not detected in human plasma samples. The exact masses of the unknown compounds were determined as 1184.519 and 800.281 (as SBD-derivatized negative ions), which possessed a product ion common to SBD-thiols (m/z 230.954, as [SBD-SH](-)) upon tandem MS spectrometric analysis.

Quantification of Biological Thiols in the Plasma of a Homocystinuria Model with Cystathionine β-Synthase Deficiency Utilizing Hydrophilic Interaction Liquid Chromatography and Fluorescence Detection

“…In our previous study, we found two unknown thiols in the plasma of 129SV mice, which were confirmed to possess an SBD-S moiety and molecular weights of 1184.519 (SBD-T1 -) and 800.281 (SBD-T2 -) by HILIC-electrospray negative ionization-mass spectrometry [14]. In this study, two peaks (peaks 7 (SBD-T1) and 8 (SBD-T2) in Fig.…”

“…Plasma samples were stored at -80°C until use. Pretreatment of plasma samples was carried out according to a previous report [14]. Briefly, MPG was added to plasma samples, and disulfides in the samples were reduced to their thiol forms by TCEP for total biothiol (=disulfide+thiol) quantification.…”

“…Recently, we developed an analytical method utilizing ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F) as a fluorogenic derivatization reagent and hydrophilic interaction liquid chromatography (HILIC) as a separation mode to tackle these problems [12][13][14]. Since HILIC is more suitable for retention and separation of highly polar compounds than RPLC, and the fluorescence of SBD-thiols is enhanced in the acetonitrile-rich mobile phase of HILIC [12,15,16], this newly developed method enables simultaneous quantification of all biological thiols described above in human and mouse plasma samples.…”

Analysis of Total Thiols in the Urine of a Cystathionine β-Synthase-Deficient Mouse Model of Homocystinuria Using Hydrophilic Interaction Chromatography

“…The most common method for determination of thiols is high-performance liquid chromatography (HPLC) [5]. In our previous studies [6,7], we utilized hydrophilic interaction chromatography (HILIC) to separate thiols in biological fluids, including human plasma and mouse serum. Pretreatment was followed by reduction with tris(2-carboxyethyl) phosphine (TCEP), deproteinization, and derivatization with ammonium 7-fluoro-2,1,3-benzoxadiazole-4-sulfonate (SBD-F).…”

Metabolomic signature of brain cancer

“…109,110 A variety of analytical methods have been developed for analysis including HPLC, GC, and CE coupled with UV and fluorescence detection. [106][107][108][111][112][113][114][115][116][117][118] These methods require pre-or post-column derivatization and involve a less sensitive, complicated, and time-consuming procedure.…”